This research is directed toward two complementary goals: understanding the regulatory mechanisms which govern the life cycle of Salmonella typhimurium temperate bacteriophage P22, and describing P22 chromosome structure. The studies outlined in this application approach three central questions. What is the physical arrangement of genes on the P22 chromosome? What are the major regulatory signals which govern the P22 life cycle? What is the molecular basis for the specificity of the P22 headful packaging mechanism for P22 DNA? Work in my laboratory has shown that investigations of these questions are closely related. The experiments proposed in this application use the information gained from these studies to approach four specific goals. 1) Prepare a physical-genetic map of the P22 chromosome. We are preparing a physical map in which the distance between any pair of EcoRI and/or HindIII cleavage sites will be proportional to the number of base pairs between the sites. When this physical map is complete, we will locate P22 genes with respect to the restriction endonuclease cleavage sites, resulting in a map in which the distances between genes on the map is proportional to physical distance between genes. 2) Determine the temporal sequence in which P22 genes function during the lytic cycle. Transcription of specific groups of P22 genes will be measured by hybridizing radioactive labeled RNA of infected cells with segments of P22 DNA carrying only a few genes. These segments will be obtained by restriction endonuclease cleavage of P22 DNA, or as P22 substitutions in lambda immP22 hybrid chromosomes. 3) Describe some mechanisms which regulate this temporal sequence of gene function. The action of major P22 regulatory genes will be defined in terms of differences in time or amount of transcription of specific groups of genes following infection by a regulatory mutant. 4) Describe functions which determine specificity of the P22 head assembly mechanism for P22 DNA. Restriction enzyme analysis and genetic techniques will be used to investigate P22 mutants which package DNA with altered specificity.